Journal: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a ) Immunoblots of Nogo-A, NgR1 and LOTUS in the olfactory bulb at E14, 15, 16, 17, 18 and P0. WT, LOTUS-KO and NgR1-KO indicate protein lysates from wild-type mice at P305, lotus -deficient mice at P237 and ngr1 -deficient mice at P91, respectively. β-actin is used as an internal control protein. ( b ) The expression levels of Nogo-A, NgR1 and LOTUS are quantified by the intensity of each protein immunoblot and normalized to the intensity of β-actin. The significance level was analyzed by performing a Kruskall-Wallis test with Dunn’s multiple comparison analysis. (Nogo-A: n = 4 experiments; LOTUS: n = 3 experiments; NgR1: n = 3 experiments).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Western Blot, Control, Expressing, Comparison

Journal: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a1,b1 ) Immunostaining of βIII-tubulin, a neuronal marker. ( a2,b2 ) Immunostaining of Tau-1, an axonal marker in the same cells of (1), ( a3,b3 ) merged images of (1) and (2), ( a4,b4 ) and immunostaining of Nogo-A in pseudo-color. ( a5,b5 ) Dashed boxes in images of a3 and b3 correspond to images of a5 and b5 at higher magnification, respectively. Arrowheads indicate axonal collateral branches. Dissociated OB neurons from E14.5 wild type mice (NgR1+/+) ( a ) or ngr1 -deficient mice (NgR1−/−) ( b ) were cultured for 7days. Branching points in ngr1 -deficient (NgR1−/−) mice were decreased in comparison with the wild type. ( c ) Quantitative analysis of axonal branching points in cultured OB neurons. Significance, indicated by (*), was obtained by performing Student’s unpaired t-test. *is P = 0.0437 (LOTUS+/+ versus LOTUS−/− (n = 4)), ** is P = 0.0029 (NgR1+/+ versus NgR1−/− (n = 3)), and comparisons indicated by (†) were obtained by performing a one-way ANOVA test with all pairwise multiple comparisons test (Tukey’s test). † is P < 0.05. †† is P < 0.000001. Scale bars: 50 μm.( d ) Quantitative analysis of axon lengths in cultured OB neurons. Axon lengths of Tau-1-positive neurites were measured using NIH ImageJ software. Nogo-A knockdown (sh-Nogo, using sh-1 in LOTUS+/+ and sh-3 in LOTUS−/−) resulted in the increase of axon length. Similarly, axon lengths in ngr1 -deficient (NgR1−/−) mice were increased compared to those in the wild-type mice. Significance was obtained by performing one-way ANOVA with Holm-Sidak’s multiple comparison test. * P = 0.0192 (control versus sh-Nogo treatment (n = 17–34 axons)); ** P = 0.0218 (wild type versus NgR1−/− (n = 4 littermates)); *** P = 0.0229 (wild type versus LOTUS−/− (n = 5 littermates)).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Immunostaining, Marker, Cell Culture, Comparison, Software, Knockdown, Control

Journal: Scientific Reports

Article Title: Axonal branching in lateral olfactory tract is promoted by Nogo signaling

doi: 10.1038/srep39586

Figure Lengend Snippet: ( a ) Schematic drawing of the lateral view of axonal branching in the LOT. The dashed square indicates the position of branch point analysis. ( b–e ) Lateral views of LOT axonal branching in whole brain from lotus -deficient mice (LOTUS−/−) and ngr1 -deficient (NgR1−/−) mice at E18. Compared to wild-type mice ( b ), the axonal branching points of the LOT were increased (white arrowheads) in LOTUS−/− ( c ), whereas the branching points in NgR1−/− were decreased ( d ). Abnormally increased axonal branching observed in LOTUS−/− was rescued in double homozygous mutants of lotus and ngr1 (LOTUS−/−; NgR1−/−) ( e ). Scale bar: 50 μm. ( f–h ) Quantitative analysis of branching points in the LOT. ( f ) Comparison of wild-type, LOTUS+/− and LOTUS−/− mice. ( g ) Comparison of wild-type, NgR1+/− and NgR1−/− mice. ( h ) Comparison of wild-type, NgR1+/− and NgR1−/− mice in the background of LOTUS−/− mice. Significance, indicated by (*), was analyzed using a Tukey’s one-way ANOVA test. ( f ) * is P = 0.031 (LOTUS+/+ versus LOTUS−/− (n = 5 littermates)). ( g ) ** is P = 0.00008 (NgR1+/+ versus NgR1−/− (n = 4 littermates)) and *** is P = 0.0016 (NgR1+/− versus NgR1−/− (n = 4 littermates)). ( h ) **** is P = 0.0001 (LOTUS−/−; NgR1+/+ versus LOUTS−/−; NgR1−/− (n = 3 littermates)) and ***** is P = 0.0076 (LOTUS−/−; NgR1+/+ versus LOTUS−/−; NgR1+/− (n = 3 littermates)).

Article Snippet: The monoclonal mouse antibody against Nogo-A (Millipore, Temecula, CA, USA), polyclonal goat antibody against mouse NgR1 (R&D Systems, Minneapolis, MN, USA), polyclonal rabbit antibody against βIII-tubulin (Genscript, Piscataway, NJ, USA), monoclonal mouse antibody against Tau-1 (Millipore), polyclonal rabbit antibody against Tau-1 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal mouse antibody against β-actin (Sigma-Aldrich, St. Louis, MO, USA), biotinylated goat antibody against hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated goat antibody against hamster IgG (Jackson ImmunoResearch), biotinylated donkey antibody against goat IgG (Jackson ImmunoResearch), Alexa488-labeled goat antibody against mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa488-labeled donkey antibody against guinea pig IgG (Jackson ImmunoResearch), Alexa594-labeled goat antibody against mouse IgG (Jackson ImmunoResearch), Alexa647-labeled goat antibody against rabbit IgG (Invitrogen), horseradish peroxidase (HRP)-conjugated donkey antibody against rabbit IgG (GE Healthcare), HRP-conjugated sheep antibody against mouse IgG (GE Healthcare, Chicago, IL, USA), HRP-conjugated donkey antibody against goat IgG (Jackson ImmunoResearch) and HRP-conjugated donkey antibody against mouse IgM (Jackson ImmunoResearch) were obtained commercially.

Techniques: Comparison

Journal: Cell reports

Article Title: Fast Regulation of GABA A R Diffusion Dynamics by Nogo-A Signaling.

doi: 10.1016/j.celrep.2019.09.015

Figure Lengend Snippet: Figure 2. Activity-Dependent Localization of Nogo-A at Synapses (A) Western blot for Nogo-A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in synaptosomes from acute hippocampal slices treated (n = 4) or not treated (n = 4) with 55 mM KCl. The graph shows quantification for the relative protein abundance. (B) Western blot (above) for GluR1 and GAPDH in synaptosomes from acute hippocampal slices with either control antibody (n = 5), 55 mM KCl (n = 5), or Nogo-A function-blocking antibody (n = 3) and quantification for the relative protein amount (below). (C) mEPSC recordings before and 10 min after the application of control or Nogo-A blocking antibody. Scale bars, 20 pA and 200 ms. (D and E) mEPSC amplitude (D) and frequency (E) percentage change upon control (black, n = 10) or Nogo-A blocking antibody (red, n = 11, ANOVA treatment, p < 0.01, F1,19 = 14.13). Values represent means ± SEMs. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: The loss-of-function for the Nogo-A signaling was achieved by application of: a monoclonal Nogo-A specific, function-blocking antibody against an 18-aa peptide within the NiG-D20 domain, the most inhibitory region of Nogo-A (mouse IgG1 11C7; 5mg / mL gift from Martin Schwab, ETH and University of Zurich; Liebscher et al., 2005; Oertle et al., 2003); an antagonist of the sphingosine-1-phosphate receptor 2 (S1PR2; 5 mM JTE-013; Tocris) or a function-blocking antibody against the Nogo receptor NgR1 (5 mg/ml affinity-purified goat IgG anti-Nogo receptor; R&D Systems).

Techniques: Activity Assay, Western Blot, Quantitative Proteomics, Control, Blocking Assay

Journal: Cell reports

Article Title: Fast Regulation of GABA A R Diffusion Dynamics by Nogo-A Signaling.

doi: 10.1016/j.celrep.2019.09.015

Figure Lengend Snippet: Figure 3. Nogo-A Signaling Promotes GABAAR Clustering at Synapses via the S1PR2 (A–D) Live-cell immunolabeling of surface GABAARs followed by immunofluorescence for synapsin in primary hippocampal neurons treated for 10 min with control (A, left) or Nogo-A blocking antibody (A, right), boiled D20 (B, left) or D20 peptide (B right), DMSO (C, left) or S1PR2 inhibitor JTE-013 (C, right), and control (D, left) or NgR1 neutralizing antibody (D, right). All of the images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values. Scale bar, 2 mm. (E–P) Normalized GABAAR cluster density, fluorescence intensity, and density of colocalized GABAAR and synapsin+ puncta upon Nogo-A loss of function (E–G, red, n = 30; Ctrl Ab, gray, n = 30), Nogo-A gain of function (H–J, red, n = 33; boiled D20 peptide, gray, n = 32), S1PR2 loss of function (K–M, green, n = 43; DMSO Ctrl, gray, n = 51), and NgR1 loss of function (N–P, blue, n = 39; Ctrl Ab, gray, n = 45). Values represent means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The loss-of-function for the Nogo-A signaling was achieved by application of: a monoclonal Nogo-A specific, function-blocking antibody against an 18-aa peptide within the NiG-D20 domain, the most inhibitory region of Nogo-A (mouse IgG1 11C7; 5mg / mL gift from Martin Schwab, ETH and University of Zurich; Liebscher et al., 2005; Oertle et al., 2003); an antagonist of the sphingosine-1-phosphate receptor 2 (S1PR2; 5 mM JTE-013; Tocris) or a function-blocking antibody against the Nogo receptor NgR1 (5 mg/ml affinity-purified goat IgG anti-Nogo receptor; R&D Systems).

Techniques: Immunolabeling, Control, Blocking Assay

Journal: Cell reports

Article Title: Fast Regulation of GABA A R Diffusion Dynamics by Nogo-A Signaling.

doi: 10.1016/j.celrep.2019.09.015

Figure Lengend Snippet: Figure 5. Nogo-A Modulates GABAAR Clustering Independently of Gephyrin (A) Immunofluorescence for gephyrin and synapsin in primary hippocampal neurons treated for 10 min with control (left) or Nogo-A blocking antibody (right). All of the images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values. Scale bars, 2 mm. (B–D) Normalized density of gephyrin clusters (B), fluorescence intensity (C), and density of gephyrin clusters colocalized with synapsin+ puncta (D, Ctrl Ab, n = 48; Nogo-A Ab, n = 50) upon treatment with control (gray, n = 63) or Nogo-A blocking antibody (red, n = 65). Values represent means ± SEMs.

Article Snippet: The loss-of-function for the Nogo-A signaling was achieved by application of: a monoclonal Nogo-A specific, function-blocking antibody against an 18-aa peptide within the NiG-D20 domain, the most inhibitory region of Nogo-A (mouse IgG1 11C7; 5mg / mL gift from Martin Schwab, ETH and University of Zurich; Liebscher et al., 2005; Oertle et al., 2003); an antagonist of the sphingosine-1-phosphate receptor 2 (S1PR2; 5 mM JTE-013; Tocris) or a function-blocking antibody against the Nogo receptor NgR1 (5 mg/ml affinity-purified goat IgG anti-Nogo receptor; R&D Systems).

Techniques: Control, Blocking Assay

Journal: Cell reports

Article Title: Fast Regulation of GABA A R Diffusion Dynamics by Nogo-A Signaling.

doi: 10.1016/j.celrep.2019.09.015

Figure Lengend Snippet: Figure 6. Nogo-A Loss of Function Increases Ca2+ Dynamics in Hippocampal Neurons to Promote GABAAR Diffusion (A) Images and relative fluorescence intensity traces for GCaMP5g expressing primary hippocampal neurons before (0 min, above) and after (20 min, below) application of Nogo-A blocking antibody. (B and C) Normalized Ca2+ transient amplitude (B) and frequency (C) over time for dendritic spines of primary hippocampal neurons treated with, from left, control (black, n = 42), Nogo-A blocking (red, ANOVA treatment, p < 0.01, F1,62 = 8.014, n = 37), NgR1 blocking antibody (blue, n = 11), control DMSO (black, n = 15), S1PR2 antagonist JTE-013 (green, ANOVA treatment, p < 0.01, F1,28 = 8.490, n = 15), boiled D20 peptide (black, n = 6), or D20 peptide (orange, ANOVA treatment, p = 0.058, F1,16 = 4.318, n = 8). (D) GABAAR-QD trajectories over 30 s and Fluo-4 fluorescence in primary hippocampal neurons. Scale bar, 5 mm. (E and F) Normalized percentage change over time for the diffusion coefficient median upon control (black, n = 12) and Nogo-A blocking antibody (red, n = 12) at synaptic (E, ANOVA treatment, p < 0.001, F1,24 = 16.25) and extrasynaptic sites (F, ANOVA treatment, p < 0.01, F1,24 = 8.006). (G) Normalized Fluo-4 fluorescence intensity change over time (F/F0) for control (black, n = 12) and Nogo-A blocking antibody (red, ANOVA treatment, p < 0.001, F1,24 = 9.804, n = 12). (H) Correlation between the peak value for the Fluo-4 fluorescence intensity change (F/F0) and the diffusion coefficient for control (black dots, n = 12) and Nogo-A blocking antibody (red dots, n = 12). (I) Hippocampal primary neurons treated with control or Nogo-A blocking antibody with or without EGTA and stained for surface GABAARs and synapsin. All of the images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values. Scale bars, 2 mm. (J–L) Normalized GABAAR cluster density (J), fluorescence intensity (K), and colocalization of GABAAR and synapsin+ puncta (L) upon Nogo-A loss of function with (Ctrl Ab, n = 32; Nogo-A Ab, n = 33) or without EGTA (Ctrl Ab, n = 30; Nogo-A Ab, n = 29). Values represent means ± SEMs. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The loss-of-function for the Nogo-A signaling was achieved by application of: a monoclonal Nogo-A specific, function-blocking antibody against an 18-aa peptide within the NiG-D20 domain, the most inhibitory region of Nogo-A (mouse IgG1 11C7; 5mg / mL gift from Martin Schwab, ETH and University of Zurich; Liebscher et al., 2005; Oertle et al., 2003); an antagonist of the sphingosine-1-phosphate receptor 2 (S1PR2; 5 mM JTE-013; Tocris) or a function-blocking antibody against the Nogo receptor NgR1 (5 mg/ml affinity-purified goat IgG anti-Nogo receptor; R&D Systems).

Techniques: Diffusion-based Assay, Expressing, Blocking Assay, Control, Staining